We propose to develop a high-throughput method to test the functional activity of cis-regulatory elements in vivo, in specific cell types. Powered by Next Generation Sequencing, a range of high-throughput methods can generate data that allow investigators to predict the locations of potential cis-regulatory elements in the genome. Collectively these methods have generated hundreds of thousands of predictions. A major problem is that there are no corresponding technologies to validate the cis-regulatory activity of these predictions in vivo. To address this problem we propose to develop a massively parallel reporter gene assay to test the activity of cis-regulatory elements and their allelic variants in vivo, in specific cell types. Our plan is to develop methods to create large libraries of cis regulatory elements in lentiviral-based reporters, and to develop methods to assay the activity of these libraries from specific populations of cells in vivo. We propose to develop this technology first to study cis-regulation in specific cell types of the brain, but the approach will generalizeto a large range of cell types from different tissues in different organisms. Successful completion of our aims will result in a high-throughput method for testing the effects of cis-regulatory polymorphism in vivo, in specific cell types. Since a large fraction of disease causing variants are thought to reside in non-coding DNA, a scalable method to test the effects of allelic variation in cis-regulatory elements would have a large impact on the field.